Fig 1. Identification of ART5 as a multicopy suppressor of flippase mutations.

(A) Suppression of growth defects by overexpressing ART5 in Cdc50p-depleted mutants carrying a mutation synthetically lethal with cdc50Δ. Cells were grown to early log phase in SGA-Ura medium, washed, and adjusted to a concentration of 2.5 × 10⁷ cells/ml. Drops of 4 μl from 5-fold serial dilutions were spotted onto a YPDA (Cdc50p-depleted) or YPGA (Cdc50p-expressed) agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1286 (P _GAL1 -CDC50 gcs1Δ), KKT116 (P _GAL1 -CDC50 fpk1Δ), and YKT1649 (P _GAL1 -CDC50 neo1–101), all carrying YEplac195 (vector), pKT1263 (pCDC50), or pKT1720 (pART5). (B) Suppression of growth defects in flippase mutants by overexpression of ART5. Cells were grown and examined as in (A), except that the P _GAL1 -CDC50 crf1Δ mutant was incubated at 25°C for 2 days. The strains used were YKT1511 (P _GAL1 -CDC50 crf1Δ), YKT1529 (P _GAL1 -CDC50 dnf1Δ crf1Δ), YKT1513 (P _GAL1 -CDC50 lem3Δ crf1Δ), and YKT1932 (P _GAL1 -NEO1), all carrying each of the plasmids in (A), except YKT1932 carried pKT1469 (pNEO1) as a positive control. (C) Suppression of the defects in membrane trafficking in flippase mutants by overexpression of ART5. Cells were grown in YPDA medium at 25°C for 14 h (Cdc50p-depleted crf1Δ) or at 30°C for 12 h (Neo1p-depleted), followed by microscopic observation of small- or middle-budded cells. The percent of cells with polarized GFP- or mRFP-Snc1p was determined (n>100) and is shown with the mean ± standard deviation of three independent experiments. Representative images are shown. The strains used were YKT1933 (P _GAL1 -CDC50 crf1Δ GFP-SNC1) and YKT1910 (P _GAL1 -NEO1 mRFP-SNC1), both carrying each of the plasmids in (A), except YKT1910 carried pKT1469 (pNEO1) as a positive control. Bar: 5 μm. (D) Failure of ART5 overexpression to suppress the alkylphosphocholine resistance and phospholipid-binding peptide sensitivity in a flippase mutant. Cells were grown to early log phase in SDA-Ura medium, washed, and adjusted to a concentration of 5.0 × 10⁶ cells/ml. Drops of 10 μl and 4 μl from 5-fold serial dilutions were spotted onto SDA-Ura containing 5.0 μg/ml miltefosine and YPDA containing 0.5 μg/ml papuamide B (pap B) or 2.0 μM duramycin agar plates, respectively, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (WT) and YKT715 (lem3Δ), both carrying YEplac195 (vector) or pKT1720 (pART5).